Tie2 is an endothelium-specific receptor tyrosine kinase (RTK) that is required for both normal embryonic vascular development and pathological angiogenesis. Tie2 is unique among RTKs in that its ligands, the Angiopoietins, have apparently opposite actions. Angiopoietin-1 (Angl) promotes vascular maturation and stabilization, in part, by preventing increases in endothelial adhesion molecule expression and vascular permeability. In contrast, Ang2 appears to inhibit Angl's vascular stabilizing effects, and in some cases Ang2 is required for angiogenesis. Although a number of Tie2-mediated signaling pathways and cellular responses have been identified in endothelial cells, these have not sufficiently explained the functional differences between Angl and Ang2. To date, however, no studies have investigated the mechanisms by which Tie2 is downregulated. Most RTKs are downregulated in a ubiquitin-dependent fashion, and in most cases this process is mediated by the E3 ubiquitin ligase c-Cbl, c-Cbl can associate directly with activated RTKs, whereupon it effects receptor ubiquitination, internalization, and subsequent degradation or recycling. Internalization and degradation are important mechanisms by which receptor function is negatively regulated. Moreover, it has been proposed that receptor internalization is required for activation of some positive signaling pathways. Preliminary studies in our lab demonstrate that Tie2 is ubiquitinated and associates with c-Cbl in endothelial cells following ligand activation. Based on these findings, we hypothesize that Angl and Ang2 differentially regulate Tie2 ubiquitination, internalization, and downregulation, and that these differences are in part responsible for the distinct functional effects of the Angiopoietins. To test this hypothesis, the Specific Aims of this proposal are to: 1) Determine the extent of Tie2 ubiquitination and its effects on Tie2 half-life and subcellular localization; 2) Determine the role of c-Cbl in Tie2 downregulation; and 3) Determine whether ubiquitination results in modulation of Tie2 signaling and function. Accomplishing these Specific Aims will provide insights into the functional differences between Angl and Ang2. Furthermore, understanding these proteins' differential effects during angiogenesis will have important implications for our ability to treat a variety of conditions, including tumor angiogenesis, diabetic retinopathy, and ischemic cardiovascular diseases.